Efficient biomolecule recycling method and system

ABSTRACT

A technique is disclosed for recapturing and recycling biomolecule reagents. The technique may be applied in a range of settings, including biopolymer synthesis, sequencing, and so forth. Biomolecule reagents such as nucleotides and oligonucleotides used to process nucleic acids, which may be marked with fluorescent tags, carry blocking agents, and so forth, are introduced to samples in a sample container. After the desired reaction occurs with some of the biomolecule reagents, such as some of the nucleotides or oligonucleotides, the effluent stream is processed to recapture unreacted biomolecule reagents. These may be separated from other reaction components, and recycled into the same or a different sample container. The recaptured biomolecule reagents may be mixed with additional biomolecule reagents prior to reintroduction to the same or different samples.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Non-Provisional of U.S. Provisional PatentApplication No. 60/897,786, entitled “Efficient Biomolecule RecyclingMethod and System,” filed Jan. 26, 2007, which is herein incorporated byreference.

BACKGROUND

The present invention relates generally to the field of synthesizing,analyzing or otherwise processing molecules such as biologically activemolecules or polymers. More particularly, the invention relates to atechnique for recapturing or recycling biomolecule reagents includingnucleotides or nucleic acids in such processes as synthesis, ligation,sequencing, and so forth.

Nucleotides and nucleic acids are processed for many purposes. Nucleicacids may be used, for example, in research, diagnostics andtherapeutics. In general, such nucleic acids may serve as probes thatspecifically bind to unique sequences of DNA or RNA present inbiological samples. This use of nucleic acids may provide for diagnosisof the risk of particular disease states based upon the presence orabsence of a particular known gene sequence associated with the diseasestate. Such nucleotides and nucleic acids (for example, in the form ofoligonucleotides) are also used for sequencing lengths or fragments ofDNA or RNA from individuals so as to determine their genomic makeup.

Nucleic acid synthesis is typically carried out in a cyclic process thatassembles a chain of nucleotides. The nucleotides are added one by onethrough a series of chemical reactions in which a particular molecule isadded to a growing nucleic acid molecule, sometimes via catalysis, untilthe desired chain is complete. The nucleotides to be added to the chainare typically washed over samples and include blocking molecules thatprevent addition of more than one nucleotide at a time in each chain.The blocking molecule is then removed, and the next desired nucleotidemay be added in the same way.

Other uses of nucleotides and oligonucleotides include geneticsequencing. Improvements are constantly being made to processes forsequencing nucleic acid segments, which may be supported on a substratein an array or microarray. In typical DNA sequencing applicationsnucleotides of the common deoxyribonucleotide types (A, T, C and G) oroligonucleotides containing deoxyribonucleotide monomers are washed overa sample that includes a template DNA to be sequenced and a primerhybridized to the template. A nucleotide or oligonucleotide may bind ata complementary site or sequence of the DNA template that is adjacent tothe primer such that the primer is elongated by enzymatically catalyzedaddition of the nucleotide or oligonucleotide to the primer. The growingprimer is detected in each cycle to determine which nucleotide oroligonucleotide has been incorporated at each site, and the nucleotidesare then de-blocked and the cycle repeated. Due to fidelity of theenzymes in specifically adding nucleotides or oligonucleotides that arecomplementary to the template and rejecting those that are not, thesequence of the template can be determined from the sequence ofnucleotides or oligonucleotides added to the primer.

In such processes, nucleotides or oligonucleotides having fluorescentdye markers, blocking molecules, and/or other moieties are typicallyexposed to the samples in a process fluid. The process fluid alsotypically contains enzymes that catalyze primer modification. Theprocess fluid is allowed to remain in contact with the sample for adesired time to permit primer modification to take place. The processfluid is then removed in a flush operation, and subsequent processingmay occur (e.g., imaging, de-blocking, and so forth). Typically,nucleotides or oligonucleotides are provided in excess amounts to favorhigh reaction yield. Thus, only a portion of the nucleotides exposed tothe samples are consumed and the remainder is flushed away unspent.Similarly, useful enzyme is flushed away after the reaction becauseunder typical conditions the enzyme, being a catalyst, is not consumed.

Current manual and automated systems for both synthesis and sequencingusing nucleotides, oligonucleotides and enzymes typically discard thesebiomolecule reagents contained in the process stream once the stream hasbeen adequately exposed to the sample. However, such molecules can becostly, and the cost of replacing these biomolecule reagents at eachcycle can become a significant cost for the overall process.

BRIEF DESCRIPTION

The present invention provides improved techniques for processingmolecules such as biopolymers that promises to be more efficient andless costly than heretofore known approaches. The techniques allow forthe recapture and eventual recycling of biomolecule reagents used inprocess fluids. The molecules may include attached fluorescent dyes,blocking moieties, or other moieties that facilitate process chemistry,or may be free of such moieties. The techniques may employ separatingone or more of the recaptured biomolecule reagents from one or moreother reaction component prior to recycling in the process. Moreover,the technique may be used in a wide range of processes, includingsynthesis, ligation, sequencing, and so forth. In certain presentlycontemplated embodiments, the recaptured biomolecule reagents may bemixed with additional biomolecule reagents before being reintroducedinto the process. These recycled molecules may then be introduced to thesame or a different sample, depending upon the design of the process andthe flow of samples through the process.

The present invention provides a method for processing a biopolymersample. The method can include a step of introducing a process fluidcontaining biomolecule reagents to a sample container in which a portionof the biomolecule reagents modify a biopolymer sample in the container;processing an effluent stream of the process fluid flowing from thesample container to recapture biomolecule reagents in the effluentstream that are present after the biopolymer sample is modified; andintroducing the recaptured biomolecule reagents into the same or adifferent sample container under conditions for modifying a biopolymersample with the recaptured biomolecule reagents.

Also provided is a system for processing a biopolymer sample. The systemcan include a sample container in which a biopolymer sample is disposed;a process fluid introduction subsystem configured to introduce a processfluid containing biomolecule reagents into the sample container in whicha portion of the biomolecule reagents modify the biopolymer sample; andan effluent stream processing subsystem configured to process theprocess fluid flowing from the sample container to recapture biomoleculereagents in the effluent stream that remain after the biopolymer sampleis modified, and to recirculate the recaptured biomolecule reagents intothe process fluid introduction subsystem for introduction into the sameor a different sample container.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a diagrammatical overview of a system for processingbiomolecule reagents, such as for synthesis or sequencing making use ofthe recapture and recycling approach of the invention;

FIG. 2 is a diagrammatical overview of an exemplary multi-stationprocessing system, such as one used for sequencing;

FIG. 3 is a diagrammatical summary of how nucleotides may be introducedto samples for sequencing the samples in an array through systems suchas those shown in FIGS. 1 and 2; and

FIG. 4 is a diagrammatical representation of the use of oligonucleotidein sequencing by ligation.

DETAILED DESCRIPTION

Turning to the drawings, and referring first to FIG. 1, an exemplaryprocessing system 10 is illustrated for processing molecules, such asnucleotides, oligonucleotides or other bioactive reagents. The systemmay be designed for synthesizing biopolymers, such as DNA chains, or forsequencing biopolymers. Exemplary biopolymers include, but are notlimited to nucleic acids such as deoxyribonucleic acid (DNA),ribonucleic acid (RNA), or analogs of DNA or RNA such as those set forthin further detail below. Other exemplary biopolymers include proteins(also referred to as polypeptides), polysaccharides or analogs thereof.Although any of a variety of biopolymers can be used, for the sake ofclarity, the systems and methods of the invention will be exemplifiedwith regard to processing nucleic acids.

In general, the illustrated system will act upon samples 12 which mayinclude one reaction site or an array of reaction sites. As used herein,the term “array” refers to a population of different reaction sites onone or more substrates such that different reaction sites can bedifferentiated from each other according to their relative location.Typically, a single species of biopolymer is attached to each individualreaction site. However, multiple copies of a particular species ofbiopolymer can be attached to a particular reaction site. The arraytaken as a whole will typically include a plurality of differentbiopolymers attached at a plurality of different sites. The reactionsites can be located at different addressable locations on the samesubstrate. Alternatively, an array can include separate substrates, suchas beads, each bearing different reaction sites. Examples of usefularrays are set forth in further detail below where nucleic acidsequencing reactions are described. The sites may include fragments ofDNA attached at specific locations in an array, or may be wells in whicha target product is to be synthesized. In a presently contemplatedembodiment, the processing system 10 may be designed for continuouslysynthesizing or sequencing molecules, particularly polymeric moleculesbased upon common nucleotides. For clarity, the system and methods ofthe invention are exemplified below with regard to particular biopolymersynthesis and sequencing reactions. Those skilled in the art will knowor be able to determine other reactions that can be used as well.

In the illustrated embodiment, system 10 includes a process fluidintroduction subsystem referred to generally by reference numeral 14,and an effluent stream processing subsystem designated generally byreference numeral 16. The samples themselves will typically be disposedin a sample container 18 through which a process fluid designatedgenerally by line 20 in FIG. 1 is introduced. In general, the processfluid will include various biomolecule reagents that are allowed toreact with biopolymers of the samples in each reaction cycle. Asdiscussed below, individual nucleotides of a single type (A, T, C or G)may be contained in the process fluid, such as for synthesizing chainsof DNA by cyclic addition of monomers. Alternatively, the process mayallow for sequencing by introduction of the four common nucleotide typesin the same process fluid such that the nucleotides attach at differentsites in each sample. Still further, polymeric biomolecule reagents,such as oligonucleotides used for synthesis by ligation or sequencing byligation, can be introduced in the process fluid. Other biomoleculereagents that can be introduced include those described below in thecontext of various sequencing or synthesis techniques. In a typicalapplication, the process fluid introduction subsystem 14 may alsodeliver a process fluid 20 that includes fluids for flushing the sample,de-blocking molecules, and so forth.

The effluent stream, designated generally by reference numeral 22, caninclude nucleotides, and oligonucleotides (if used in the processstream) that did not react with the sample during a reaction cycle. Ingeneral, the process fluid introduced for such reactions will includemany times more nucleotides and/or oligonucleotides than are requiredfor the synthesis or sequencing process. Additionally or alternatively,the effluent stream can include an enzyme, such as a polymerase orligase, that remains active following the reaction cycle. The effluentstream may be drained from the samples in various manners, but in apresently contemplated embodiment, the stream is created by control ofvalving (not shown) that allows the flow of fluids from the container orcontainers 18 after sufficient time has elapsed for the reaction cycle.The process fluid may be forced through the system by one or more pumps,as indicated by reference numeral 24 for a pump in a process fluidintroduction subsystem 14. The pump will typically draw process fluidsfrom a source 26 where biomolecule reagents such as nucleotides andenzymes introduced into the system, as indicated by reference numeral28, are mixed with other fluids and reagents. Other pumps andappropriate conduits, valving, and so forth (not shown) will typicallybe provided for other fluids and reagents, such as those used forflushing the sample container after a reaction cycle, de-blockingnucleotides and oligonucleotides, and so forth.

As will be appreciated by those skilled in the art, biomolecule reagents28 added to the process stream during any particular cycle of operationmay include only one of the common DNA nucleotides, such as whereoligonucleotides of specific sequences are to be synthesized. Forsequencing, on the other hand, all four common nucleotides may beincluded in the process stream introduced to the samples during eachcycle of operation. Again, depending upon the nature of the process,these nucleotides may be tagged with fluorescent markers such that theycan be easily imaged during each cycle of operation. In other systems,the biomolecule reagents 28 will be replaced by oligonucleotides whichwill be introduced to the samples, such as for ligation. In suchprocesses, the introduced oligonucleotides will typically be tagged forfluorescent imaging. Other biomolecule reagents 28 that can be added areset forth below in the context of different synthesis and sequencingtechniques. Those skilled in the art will know or be able to readilydetermine various combinations of the reagents based on the conceptsexemplified for nucleic acid processing and that which is describedbelow or otherwise known for the different synthesis and sequencingtechniques.

The effluent stream processing subsystem 16 may include a separationsystem 30 that isolates the nucleotides, enzymes and/or otherbiomolecule reagents from certain other constituents and reagents of theeffluent stream. Several different types of separation methods may beenvisaged for this purpose, such as filtration; solid-phase extraction;liquid phase extraction; chromatographic techniques such as sizeexclusion chromatography (SEC), ion exchange chromatography, reversephase chromatography, normal phase (silica) chromatography, affinitychromatography; electrophoresis or the like. A method appropriate forthe particular biomolecule reagent can be used. If desired, the methodcan be specific for only a subset of the biomolecule reagents to berecycled. For example, the method can separate nucleotides from enzymesor can even separate different species of nucleotides from each other.However, it is also possible to use a method that co-isolates all fourspecies of nucleotides together or co-isolates nucleotides and enzymes.

The separation system 30 can further function to concentrate abiomolecule reagent that is separated from the effluent stream.Concentration can be a byproduct of the particular separation methodused. For example, SEC and solid phase extraction typically have theeffect of eluting biomolecule reagents in a more concentrated form. Ifdesired, the separation system can include a subsystem that is capableof concentrating a biomolecule reagent of interest prior to recycling ora separate concentration step can be included prior to recycling.Concentration can be carried out by SEC, solid phase extraction,precipitation, and/or removal of solvent by evaporation or other knownmethod.

The separation system 30 may output two separate process streams, oneincluding recaptured enzymes, nucleotides or oligonucleotides, and asecond waste stream which is forwarded for waste disposal as indicatedby reference numeral 32 in FIG. 1. Such fluids may be disposed of in anyconventional manner. The recaptured nucleotides or oligonucleotides maybe mixed with additional nucleotides or oligonucleotides as indicatedgenerally by reference numeral 34 in FIG. 1. Depending upon the volumeor flow rate desired for the system, and the amount and type ofnucleotides or oligonucleotides recaptured by the system, and thepercentage balances of recaptured material and additional material maybe controlled by the mixing system 34. This material may then beadvanced to the process fluid source 26 for reintroduction to the samesample or sample container, or to different samples or different samplecontainers.

A detection system 35 can be included in the system of FIG. 1 to allowquantitation of enzymes, nucleotides, or oligonucleotides that are to berecycled. The detection system can be placed to detect biomoleculereagents that are separated by the separation system 30 prior todelivery of the reagents to the mixing system 34 (or prior to deliveryto the process fluid source 26 in embodiments that do not include themixing system 34). Detection can be via any technique that isappropriate to quantitate a distinguishing property of a biomoleculereagent of interest. Exemplary properties upon which detection can bebased include, but are not limited to, mass, electrical conductivity,energy absorbance, fluorescence, magnetism, luminescence, or the like.For example, nucleotides, oligonucleotides, or enzymes having afluorescent label can be quantified using a UV/VIS detector orfluorimeter that detects light absorbance or emission in a wavelengththat is specific to the label. Other detection techniques that can beused include, for example, mass spectrometry which can be used toperceive a biomolecule reagent based on its mass; surface plasmonresonance which can be used to perceive a biomolecule reagent based onbinding or dissociation from a surface; electrical conductance orimpedance which can be used to perceive a biomolecule reagent based onchanges in its electrical properties or in the electrical properties ofits environment; magnetic resonance which can be used to perceive abiomolecule reagent based on presence of magnetic nuclei; or other knownanalytic spectroscopic or chromatographic techniques.

The detection system 35 can interface with a computer that is capable ofprocessing detected signals to determine the quantity of a particularrecaptured biomolecule reagent of interest. The computer can further beprogrammed to determine an appropriate amount of additional biomoleculereagent to add to the recaptured biomolecule reagent at mixing system 34in order to provide a desired quantity of biomolecule reagent for thenext cycle of the sequencing or synthesis reaction being carried out. Inthis way recycling can be carried out in a fully automated fashionwithout intervention of a human user for one or more cycles of asequencing or synthesis reaction. Alternatively or additionally, thesystem can be configured to report the quality or quantity of therecaptured biomolecule reagent to a human user.

FIG. 2 is a diagrammatical overview of a biopolymer processing system 36that may employ a biomolecule reagent delivery system 10 of the typediscussed with reference to FIG. 1. In general, system 36 may include aplurality of stations through which samples in sample containers 18progress. The system may be designed for cyclic operation in whichreactions are promoted with single nucleotides or with oligonucleotides,followed by flushing, imaging and de-blocking in preparation for asubsequent cycle. In a practical system, the samples 18 may becirculated through a closed loop path for sequencing, synthesis orligation.

In the illustrated embodiment, the nucleotide delivery system 10provides process stream 20 to a sample container 18. As discussed withreference to FIG. 1, then, the effluent stream 22 from the container isrecaptured and recirculated in the nucleotide delivery system, forrecapture of enzymes, nucleotides and oligonucleotides (where used) fromthe effluent stream. These are recycled, such as with additionalenzymes, nucleotides or oligonucleotides being added, as discussed abovewith reference to FIG. 1. In the illustrated embodiment, then, thesample container may be flushed at a flush station 38 to removeadditional reagents and to clarify the sample for imaging. The sample isthen moved to an imaging system 40 where image data may be generatedthat can be analyzed for determination of the sequence of aprogressively building oligonucleotide chain, such as based upon a knowntemplate as described below. In a presently contemplated embodiment, forexample, imaging system 40 may employ confocal line scanning to produceprogressive pixilated image data that can be analyzed to locateindividual sites in an array and to determine the type of nucleotidethat was most recently attached or bound to each site. Following imagingstation 40, then, the samples may progress to a de-blocking station 42in which a blocking molecule or protecting group is cleaved from thelast added nucleotide, along with the marking dye.

In a typical sequencing system, then, image data from the imaging system40 will be stored and forwarded to a data analysis system as indicatedgenerally at reference numeral 44. The analysis system will typicallyinclude a general purpose or application-specific programmed computerproviding for user interface and automated or semi-automated analysis ofthe image data to determine which of the four common DNA nucleotides waslast added at each of the sites in an array of each sample. As will beappreciated by those skilled in the art, such analysis is typicallyperformed based upon the color of unique tagging dyes for each of thefour common DNA nucleotides. However, tags having other distinguishingproperties, whether detectable by imaging or any other useful method,can be used if desired including, for example, tags having thoseproperties set forth above in regard to the detection system of FIG. 1.This image data is further analyzed by a sequencing system 46 which mayderive sequence data from the image data, and piece together sequencedata for a multitude of oligonucleotides or DNA fragments to providemore comprehensive genomic mapping of a particular individual orpopulation.

FIG. 3 illustrates a typical reaction cycle in a sequencing by synthesistechnique for oligonucleotides that may benefit from the nucleotiderecapture and recycling technique of the present invention. In general,the synthesis operation summarized in FIG. 3 may be performed on asample 12 comprising a support 48 on which a multitude of sites 50 and52 are formed. In the preparation of each sample 12, many such sites maybe formed, each with unique fragments of genetic material as indicatedgenerally by reference numeral 54. These fragments may constitutetemplates of DNA or RNA to be sequenced. The fragments can be isolatedfrom a biological source using methods known in the art. In embodimentsutilizing amplification methods, the fragments can be amplicons of a DNAor RNA isolated from a biological source. Each template comprises anumber of mers or bases 56 which will uniquely bind to a complimentarynucleotide (or analog thereof) during the synthesis process. Thesequencing process begins with binding of an anchor primer 58 to each ofthe templates. This anchor primer includes complimentary bases 60 thatbind with those of a corresponding sequence of the template. Theremaining portion of the template, designated generally by referencenumeral 62, constitutes that portion to be sequenced. The length 64 ofthis portion may vary, with presently contemplated embodiments extendingfrom 25 to 40 bases or more.

As sequencing progresses, the introduced processed stream will includeall four common DNA nucleotides, one of which will add to the primer ata position that is opposite the next available base in the template, asindicated by reference numeral 66. The added nucleotide will include abase 68 that is complementary to the template as well as a fluorescenttag 70 and a blocking molecule 72. As will be noted by those skilled inthe art, as used herein, the term “nucleotides” in the illustratedprocesses will typically include units from which DNA molecules areconstructed. Although any nucleotides or oligonucleotides may berecaptured and recycled in accordance with the present technique, inmany practical applications, these will includedeoxynucleotide-triphosphates (dNTP), each carrying a single nitrogenousbase (adenine, guanine, cytosine or thymine). The complimentarynucleotide is added to the primer due to the activity of a polymerase,as indicated generally by reference numeral 74. Other nucleotides thanthe specific one binding to the template will also be present in theprocess fluid, as indicated generally by reference numerals 76, 78 and80 in FIG. 3. Nucleotides not binding to the templates will subsequentlybe washed from the sample in a flushing operation, exiting in theeffluent stream to be recaptured and recycled as described above.

FIG. 4 is a diagrammatical representation of a sequencing by ligationreaction. As in the case of FIG. 3, the sample 12 may include sitesformed on a support 48. Each site may include a unique fragment ofgenetic material or a template 54 to be sequenced. The ligation processalso begins with binding of an anchor primer 58 to the template.However, in the ligation reaction, query probes 82, each carrying aunique fluorescent tag 70 are introduced to the sample in a processstream. The primers may bind to a query position on the template and beadded to the primer in the presence of ligase as indicated generally byreference numeral 84. In general, each of the query probes will includea single dNTP base which attaches to a corresponding location on thetemplate. As in the previous example, the oligonucleotides comprisingthe query primers are then washed from the sample in a flushingoperation, recaptured and recycled as described above. The sequencing byligation reaction can include a step of removing part of the sequence ofthe added probe using a restriction endonuclease as set forth in furtherdetail below. The restriction endonuclease can also be recycled usingmethods set forth herein. There is a variety of nucleic acid sequencingmethods that can be used in accordance with the methods set forthherein, most of which involve cycle sequencing consisting of repeatedrounds of sequencing biochemistry interspersed by imaging. Severalformats of cycle sequencing have been described in the literature, andinclude sequencing-by-synthesis (SBS), sequencing-by-ligation (SBL), andsequencing-by-hybridization (SBH). One of the most useful forms of cyclesequencing is SBS, an embodiment of which has been described above inregard to FIG. 3. In SBS the sequence of a target nucleic acid is readby repeated rounds of polymerase-based nucleotide insertion andfluorescent/chemiluminescent readout. SBS has two formats: (1) stepwisenucleotide addition (SNA) employing cycles of dNTP incorporation andimaging, and (2) cyclic reversible termination (CRT) employing cycles ofincorporation of reversible terminators, imaging, and deprotection.

The SNA approach to cycle sequencing has been described by at leastthree different groups. In one commercial implementation from 454Lifesciences, (Branford, Conn.) and Roche Diagnostics (Basel,Switzerland), cyclic pyrosequencing from assembled clonal beads has beenused to sequence entire microbial genomes. Other examples of SNA includethose that use cyclic addition of cleavable fluorescently-labeled dNTPsto sequence polony clones. After each base addition and imaging step,fluorescent labels are cleaved by disulfide reduction. In a thirdapproach, single target molecules are immobilized onto a glassmicroscope slide at a sparse density and cycle sequencing is performedby basewise addition of Bodipy-labeled dNTPs. After imaging, thefluorescence was destroyed by photobleaching.

In CRT, cycle sequencing is accomplished by stepwise addition ofreversible terminator nucleotides containing a cleavable orphotobleachable dye label. This approach is being commercialized bySolexa (www.solexa.com). The availability of fluorescently-labeledterminators in which both the termination can be reversed and thefluorescent label cleaved is important to facilitating efficient CRT.Polymerases can also be co-engineered to efficiently incorporate andextend from these modified nucleotides. In particular embodiments,reversible terminators/cleavable fluors can include fluor linked to theribose moiety via a 3′ ester linkage. Other approaches have separatedthe terminator chemistry from the cleavage of the fluorescence label.For example, reversible terminators are known that include a small 3′allyl group that blocks extension until it is deblocked by a shorttreatment with a palladium catalyst. The fluorophore can be attached tothe base via a photocleavable linker that can be cleaved by exposure tolong wavelength UV light. Thus, both disulfide reduction orphotocleavage can be used as a cleavable linker.

Another approach to reversible termination is the use of naturaltermination that ensues after placement of a bulky dye on a dNTP. Thepresence of a charged bulky dye on the dNTP can act as an effectiveterminator through steric and/or electrostatic hindrance. The presenceof one incorporation event prevents further incorporations unless thedye is removed. Cleavage of the dye removes the fluor and effectivelyreverses the termination.

The biomolecule reagents used in the above-described sequencing methodscan be recycled using a method or system of the invention. Based on theprinciples and examples set forth herein, those skilled in the art willbe able to readily apply the methods and systems of the invention todifferent sequencing techniques.

Oligonucleotide synthesis is a cyclical process that assembles a chainof nucleotides. Nucleotides are added one by one through a cycle ofchemical reactions, in which a particular molecule (e.g., a nucleotide)is added to a growing DNA molecule (e.g., a growing DNA chain),sometimes via catalysis, until the desired chain is complete. Generally,each cycle of chemical reactions includes the steps of detritylation,coupling, capping and oxidation. During the detritylation or“deprotection” step, a dimethoxytrityl (DMT) group is removed from thelast nucleotide of the growing DNA chain to allow the addition of thenext nucleotide. The amount of DMT released from each cycle is monitoredto determine coupling efficiency. The release of DMT is apparent becausea bright orange color is emitted as DMT is released. In accordance withthe systems and methods set forth herein, unused nucleotides that arepresent after individual synthetic cycles can be recycled for use in asubsequent cycle.

Any of a variety of nucleic acids can be synthesized or sequenced usingthe invention including, for example, those having a native structure orthose having an analog structure. A nucleic acid with a native structuregenerally has a backbone containing phosphodiester bonds and can be, forexample, deoxyribonucleic acid or ribonucleic acid. An analog structurecan have an alternate backbone including, without limitation,phosphoramide, phosphorothioate, phosphorodithioate,O-methylphosphoroamidite linkages, or peptide nucleic acid backbones andlinkages. Other analog structures include those with positive backbones,non-ionic backbones, and non-ribose backbones. Analog structurescontaining one or more carbocyclic sugars are also useful.

A nucleic acid useful in the invention can contain a non-natural sugarmoiety in the backbone. Exemplary sugar modifications include, but arenot limited to, 2′ modifications such as addition of halogen, alkyl,substituted alkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SO₂CH₃, OSO₂,SO₃, CH₃, ONO₂, NO₂, N₃, NH₂, substituted silyl, and the like. Similarmodifications can also be made at other positions on the sugar,particularly the 3′ position of the sugar on the 3′ terminal nucleotideor in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide. Such modifications provide useful moieties for blockingfurther extension of nucleic acids that have incorporated the analogs.Such moieties are referred to as “blocking groups.” Nucleic acids,nucleoside analogs or nucleotide analogs having sugar modifications canbe further modified to include a reversible blocking group, peptidelinked label or both.

A nucleic acid used in the invention can also include native ornon-native bases. In this regard a native deoxyribonucleic acid can haveone or more bases selected from the group consisting of adenine,thymine, cytosine or guanine and a ribonucleic acid can have one or morebases selected from the group consisting of uracil, adenine, cytosine orguanine. Exemplary non-native bases that can be included in a nucleicacid, whether having a native backbone or analog structure, include,without limitation, inosine, xathanine, hypoxathanine, isocytosine,isoguanine, 5-methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine,6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine,2-thioLiracil, 2-thiothymine, 2-thiocytosine, 15-halouracil,15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil,6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine orguanine, 8-amino adenine or guanine, 8-thiol adenine or guanine,8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halosubstituted uracil or cytosine, 7-methylguanine, 7-methyladenine,8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,3-deazaguanine, 3-deazaadenine or the like. A particular embodiment canutilize isocytosine and isoguanine in a nucleic acid in order to reducenon-specific hybridization, as generally described in U.S. Pat. No.5,681,702. A non-native base used in a nucleic acid of the invention canhave universal base pairing activity, wherein it is capable of basepairing with any other naturally occurring base. Exemplary bases havinguniversal base pairing activity include 3-nitropyrrole and5-nitroindole. Other bases that can be used include those that have basepairing activity with a subset of the naturally occurring bases such asinosine, which basepairs with cytosine, adenine or uracil.

Nucleotide precursors for the above-described nucleic acids and analogsare known in the art and can be used in the systems or methods set forthherein. Furthermore, primers or other oligonucleotides used forsequencing or synthesizing nucleic acids or nucleic acids can includeone or more of the above-described structures or biological activities.

Although the methods and systems of the invention have been exemplifiedabove with regard to nucleic acids for clarity, it will be understoodthat any of a variety of biopolymers can be synthesized, analyzed orotherwise processed using a system or method disclosed herein.Embodiments that are directed to peptide synthesis can be carried out torecapture and recycled biomolecule reagents used in the method set forthbelow.

The process of peptide synthesis on solid supports generally involvesbuilding a peptide from the carboxyl-terminal end. The peptide isattached to a solid support via its carboxy-terminal amino acid andfurther includes a protecting group on the amino-terminal α-amino group.The protecting group is then cleaved off of the peptide to form adeprotected peptide. Next, a monomeric amino acid, also containing anα-amino protecting group, is contacted with the de-protected peptideunder conditions for formation of a peptide bond between the α-aminogroup of the deprotected peptide and the α-carboxy group the monomericamino acid. The monomeric amino acid can be provided in an activatedform or an activating reagent can be added to the amino acid and growingpeptide. Washes can be carried out between steps to remove reagents. Thecycle of deprotecting the prior amino acid and coupling the additionalamino acid can be repeated until a peptide of the desired length issynthesized. Any reactive side chains of the amino acids are typicallyprotected by chemical groups that can withstand the coupling and α-aminodeprotection procedure. These side chain protecting groups, however, canbe removed at the end of the synthesis.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

1. A method for processing a biopolymer sample comprising: introducing aprocess fluid containing biomolecule reagents to a sample container inwhich a portion of the biomolecule reagents modify a biopolymer samplein the container; processing an effluent stream of the process fluidflowing from the sample container to recapture biomolecule reagents inthe effluent stream that are present after the biopolymer sample ismodified; and introducing the recaptured biomolecule reagents into thesame or a different sample container under conditions for modifying abiopolymer sample with the recaptured biomolecule reagents.
 2. Themethod of claim 1, wherein the biomolecule reagents comprise nucleotidesand the biopolymer sample comprises a nucleic acid.
 3. The method ofclaim 2, wherein the biomolecule reagents further comprise a polymeraseand the modifying of the biopolymer comprises adding at least one of thenucleotides to the nucleic acid.
 4. The method of claim 2, wherein thenucleotides comprise a single nucleotide type.
 5. The method of claim 2,wherein the nucleotides comprise blocking agents inhibiting addition ofmore than one nucleotide to a nucleic acid.
 6. The method of claim 5,wherein the blocking agents comprise fluorescent tags permitting theblocking agents to be detected in an effluent stream.
 7. The method ofclaim 2, wherein the nucleotides comprise fluorescent tags permittingdetection of the nucleotides.
 8. The method of claim 2, wherein thebiomolecule reagents comprise an oligonucleotide and the biopolymersample comprises a nucleic acid.
 9. The method of claim 8, wherein thebiomolecule reagents further comprise a ligase and the modifying of thebiopolymer sample comprises ligating the oligonucleotide to the nucleicacid.
 10. The method of claim 1, comprising separating the biomoleculereagents from other components of the effluent stream prior tointroducing the recaptured nucleotides into the same or different samplecontainer.
 11. The method of claim 1, comprising mixing the recapturedbiomolecule reagents with additional biomolecule reagents beforeintroducing the recaptured nucleotides into the same or different samplecontainer.
 12. The method of claim 11, wherein the step of introducingthe recaptured biomolecule reagents into the same or a different samplecontainer comprises introducing the mixed biomolecule reagents into thesame or a different sample container.
 13. The method of claim 1, whereinthe biomolecule reagents comprise a biomolecule reactant and themodifying of the biopolymer comprises adding the biomolecule reagent tothe biopolymer sample.
 14. The method of claim 1, wherein thebiomolecule reagents comprise an enzyme that catalyzes the modifying ofthe biopolymer.
 15. The method of claim 1, wherein the biopolymer sampleis retained in the container by attachment to a solid phase substrate.16. The method of claim 1, wherein a plurality of different biopolymersamples is present in the container and the biomolecule reagents modifythe different biopolymer samples.
 17. The method of claim 16, whereinthe plurality of different biopolymer samples comprises at least 1,000different biopolymers attached to individual sites of an array.
 18. Asystem for processing a biopolymer sample comprising: a sample containerin which a biopolymer sample is disposed; a process fluid introductionsubsystem configured to introduce a process fluid containing biomoleculereagents into the sample container in which a portion of the biomoleculereagents modify the biopolymer sample; and an effluent stream processingsubsystem configured to process the process fluid flowing from thesample container to recapture biomolecule reagents in the effluentstream that remain after the biopolymer sample is modified, and torecirculate the recaptured biomolecule reagents into the process fluidintroduction subsystem for introduction into the same or a differentsample container.
 19. The system of claim 18, wherein the system isconfigured to mix the recaptured biomolecule reagents with additionalbiomolecule reagents prior to the introduction into the same ordifferent sample container.
 20. The system of claim 18, comprising aseparation subsystem configured to separate the biomolecule reagentsfrom other components of the effluent stream prior to the introductioninto the same or different sample container.
 21. The system of claim 18,wherein the system is configured to determine the sequence of thebiopolymer sample.
 22. The system of claim 21, wherein the biopolymersample comprises a nucleic acid.
 23. The system of claim 22, wherein thebiomolecule reagents comprise nucleotides or oligonucleotides.
 24. Thesystem of claim 22, wherein the biomolecule reagents comprise apolymerase or ligase.
 25. The system of claim 18, wherein the system isconfigured to synthesize a biopolymer.
 26. The system of claim 25,wherein the biopolymer comprises a nucleic acid.
 27. The system of claim18, wherein the system is configured to quantify a biomolecule reagentin the effluent stream.
 28. The system of claim 18, wherein the systemis configured to retain the biopolymer sample in the container byattachment to a solid phase substrate.
 29. The system of claim 18,wherein the system is configured to process a plurality of differentbiopolymer samples, whereby a plurality of different biopolymer samplesare disposed in the container, and whereby the process fluidintroduction subsystem is configured to introduce a process fluidcontaining biomolecule reagents into the sample container in which aportion of the biomolecule reagents modify the plurality of differentbiopolymer samples.
 30. The system of claim 29, wherein the plurality ofdifferent biopolymer samples comprises at least 1,000 differentbiopolymers attached to individual sites of an array.